Long-acting peptide analogs

ABSTRACT

Long-acting agonistic analogs for CLR/RAMP receptors are provided that have an extended half-live in vivo.

FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT

This invention was made with Government support under contract DK070652awarded by the National Institutes of Health. The Government has certainrights in the invention.

INTRODUCTION

Many aspects of physiology, including hunger, stress responses, andreproduction, are dependent on hormone balance for control. This balancecan be responsive to both internal and external stimuli. For example,secretion of hormones by the anterior pituitary gland is controlledlargely by the hypothalamus, a region of the brain that lies just abovethe gland. Hypothalamic neurons are known to make and release peptidefactors that stimulate or inhibit the secretion of a particular hormoneby the specific set of cells that produces it in the pituitary.

Diverse hypothalamic releasing peptides are important in the regulationof the secretion of different anterior pituitary hormones such as GH,ACTH, TSH, LH, and FSH. However, the regulation of prolactin release bythe anterior pituitary is more complex, and involves stimulatory factorsoriginating from both the hypothalamus and the intermediate lobe (seeLaudon et al. (1990) Endocrinology 126:3185-3192; Ben-Jonathan andHnasko (2001) Endocr. Rev. 22:724-763). Although the role of theintermediate lobe in the regulation of prolactin secretion is welldocumented, and the intermediate and posterior lobes are necessary forthe suckling- and estradiol-induced rises in prolactin release, theidentity of prolactin-releasing factors from the intermediate loberemains to be characterized (Allen et al. (1995) Endocrinology136:3093-3099).

The pituitary calcitonin receptor-like receptor (CRLR) has beenassociated with prolactin release (Meeran et al. 1997. J. Clin.Endocrinol. Metab. 82:95-100), although there is a lack of theoverlapping calcitonin gene-related peptide (CGRP) expression patternwith binding sites for CGRPs in the brain (Kruger 1988. Brain Res.463:223-244). Originally isolated as a polypeptide hormone essential forcalcium balance, calcitonin belongs to a group of peptide hormonesincluding α CGRP, β CGRP, adrenomedullin (ADM), and amylin (Eto (2001)Peptides 22:1693-1711). These tissue-specific peptides are importantendocrine and neurocrine integrators for homeostasis maintenance in thevascular and respiratory systems.

The biological actions of these peptides are mediated via binding to twoclosely related type II G protein-coupled receptors (GPCRs), thecalcitonin receptor and the CRLR (Christopoulos et al. (1999) Mol.Pharmacol. 56:235-242; Poyner et al. (2002) Pharmacol. Rev. 54:233-246).Although the calcitonin receptor is the main mediator for calcitoninaction, it also binds amylin. Recent cloning and functional studies haveshown that CGRPs, ADM, and to a lesser extent, amylin, interact withdifferent combinations of CRLR and three receptor activity modifyingproteins (RAMPs); see McLatchie et al. (1998) Nature 393:333-339.

Many cells express multiple RAMPs. Co-expression of the calcitoninreceptor-like receptor (CRLR) and receptor activity-modifying proteins(RAMPs) is required to generate functional receptors for calcitoningene-related peptide (CGRP) and adrenomedullin (ADM). The formation ofheterodimers between RAMPs and CRLR is essential for the proper cellsurface targeting and pharmacological characteristics of both CGRP andADM receptors. The RAMP family comprises three members (RAMP1, -2, and-3), which share less than 30% sequence identity but a commontopological organization. They are small intrinsic membrane proteins(predicted sizes: M_(r) 14,000-17,000) with a large extracellular Nterminus (˜100 amino acids), a single transmembrane domain, and a veryshort intracellular domain (10 amino acids). Co-expression of RAMP1 withCRLR leads to the formation of a CGRP receptor, whereas RAMP2 and RAMP3promote the expression of an ADM receptor. When the calcitonin receptoris co-expressed with RAMP1 it provides for a CGRP/amylin receptor, andwith RAMP3 it provides for an amylin receptor.

Studies using mutant mice deficient for α CGRP, ADM, or amylin haveindicated that, in different systems, CRLR can important forcardiovascular morphogenesis, sensory neurotransmission, inflammatoryreactions, nociceptive behavior, and glucose homeostasis. Thus, thephysiological functions of peptides in this family are determined byreceptor-binding specificity and the tissue expression profiles ofindividual ligands.

Peptide hormones are of great interest for clinical use and thedevelopment of therapies, including treatment of hypertension andmaintenance of cardiovascular homeostasis. In addition to these effects,identification of prolcatin releasing factor is of interest. Althoughprolactin is important in pregnancy and lactation in mammals, and isinvolved in the development of the mammary glands and the promotion ofmilk synthesis, a specific prolactin-releasing hormone has hithertoremained unknown.

RELATED PUBLICATIONS

Hay and Smith (2001) Trends Pharmacol. Sci. 22:57-59; and Shindo et al.(2001) Circulation 104:1964-197 discuss the importance of adrenomedullinin the vasculature. The role of a CGRP is discussed by Zhang et al.(2001) Pain 89:265-273; Salmon et al. (1999) Neuroreport 10:849-854; andSalmon et al. (2001) Nat. Neurosci. 4:357-358. The role of amylin isdiscussed by Mulder et al. (2000) Am. J. Physiol. Endocrinol. Metab.278:E684-691.

GenBank entry AF529213.

SUMMARY OF THE INVENTION

Long-acting peptide analogs are provided herein, which provide for thebiological activities of intermedin, or of adrenomedullin, includingacting as ligand for the calcitonin receptor-like receptor, but whichprovide for a substantially longer in vivo half-life when compared tothe native polypeptide. Analogs of the invention provide for an in vivoeffectiveness that lasts at least 2-fold longer in duration than thenative peptide, and may be 5-fold longer, 10-fold longer, 20-foldlonger, or more. The increased in vivo activity may be measured in vivoor in vitro, by determining the stability of the polypeptide, the lengthof the physiological effect, and the like.

In some embodiments of the invention, the analogs comprise abiologically active intermedin or adrenomedullin polypeptide that hasbeen modified at the N-terminus. N-terminal modifications of interestinclude conjugation to a fatty acid, usually a C4 to C30 fatty acid,which may be unsaturated or saturated. Fatty acids of interest include,without limitation, palmitic acid; stearic acid; arachidic acid; lauricacid; myristic acid; myristoleic acid; palmitoleic acid; sapienic acid;oleic acid; linoleic acid; α-linolenic acid; arachidonic acid;eicosapentaenoic acid; erucic acid; docosahexaenoic acid; etc. In otherembodiments the polypeptide is modified by pegylation, glycosylation,conjugation to large proteins such as albumin, or conjugation withpolymers in combination with amino acid modifications such as the use ofD-amino acid or beta amino acids to increase the biological half-life.

The analogs of the invention provide a long-acting agonistic analogs forCLR/RAMP receptors. The analogs may provide limited effect on heart ratewhile effecting blood pressure significantly. The compartmentalizationof the long-acting peptide in serum effectively decreased the availablefraction of agonists for cell stimulation at a give time point, therebyalleviating the peak and trough effects of injected peptides andeliminating unwanted side effects.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1C are examples of blood pressure measurements in SHR ratsafter treatment with a long-acting intermedin peptide (Palmiticacid-modified IMD, IMD-PA), A. 30 nmoles/kg BW; B/C, 100 nmoles/kg BW.

FIGS. 2A-2B are examples of blood pressure measurements in SHR ratesafter injection with a wild type intermedin peptide (IMD, 100 nmoles/kgBW). The hypotensive effects of the IMD-PA peptide lasted over fivehours in vivo, whereas that of the unmodified IMD peptide lasted lessthan 1 hr.

FIGS. 3A-3B are BP measurements after injection with PBS in controlanimals.

FIG. 4 shows the stimulatory effect of IMD and IMD-PA in 293T cellsexpressing three different receptors (CLR/RAMP1, CLR/RAMP2, andCLR/RAMP3).

FIGS. 5A-5C show examples of systolic pressure measurement of SHR ratsafter an injection of a long-acting adrenomedullin peptide (Palmiticacid-conjugated ADM, ADM-PA, 100 nmoles/kg BW). The hypotensive effectof this peptide lasted over five hours in vivo. By contrast, thehypotensive effect of unmodified ADM peptide normally lasts less than1.5 hr.

FIGS. 6A-6B show examples of systolic pressure measurement of SHR ratsafter an injection of control saline.

FIG. 7 shows the stimulatory effect of ADM and ADM-PA in 293T cellsexpressing recombinant adrenomedullin receptor 1 (CLR/RAMP2) and 2(CLR/RAMP3). Although the stimulatory effect of ADM-PA peptide is lowerthan that of the wild type ADM in vitro, it has a significantly longereffective life in vivo.

FIG. 8 shows that a significantly higher level of immunoreactive ADM-PAis retained in vivo as compared to the ADM peptide at 12 hr after i.p.injection.

DESCRIPTION OF THE SPECIFIC EMBODIMENTS

The invention provides novel polypeptide analogs of intermedin oradrenomedullin, which are members of the calcitonin peptide hormonefamily.

Intermedin is a ligand for the calcitonin receptor-like receptor. Thehuman intermedin gene encodes a 148-amino-acid open reading frame, witha 24-amino-acid signal peptide for secretion at the N-terminus and amature amidated peptide (shown in SEQ ID NO:1 and SEQ ID NO:2). Maturehuman intermedin peptides include without limitation a 40 amino-acidpeptide (intermedin-short or IMDS), set forth as SEQ ID NO:3, whichcorresponds to residues 8-47 of the mature protein; and a 47-amino-acidmature peptide (intermedin-long or IMDL), set forth as SEQ ID NO:4. Theintermedin peptide may be substituted with a terminal lysine residue forease of modification. For example, an intermedin peptide of interest isas set forth in SEQ ID NO:7:K(mod)GCVLGTCQVQNLSHRLWQLMGPAGRQDSAPVDPSSPHSY, where the terminal lysineis modified, e.g. by attachment of a lipid or other group.

Adrenomedullin is a ligand for the calcitonin receptor-like receptor.The ADM gene encodes for a preprohormone, which is posttranslationallymodified to generate 2 biologically active peptides: adrenomedullin andproadrenomedullin N-terminal 20 peptide (PAMP). Adrenomedullin consistsof 52 amino acids, has 1 intramolecular disulfide bond, and shows slighthomology with the calcitonin gene-related peptide. The precursor, calledpreproadrenomedullin, is 185 amino acids long. See Genbank referenceNM_001124, herein specifically incorporated by reference. The precursorpolypeptide (SEQ ID NO:5) has the amino acid sequence:

MKLVSVALMYLGSLAFLGADTARLDVASEFRKKWNKWALSRGKRELRMSSSYPTGLADVKAGPAQTLIRPQDMKGASRSPEDSSPDAARIRVKRYRQSMNNFQGLRSFGCRFGTCTVQKLAHQIYQFTDKDKDNVAPRSKISPQGYGRRRRRSLPEAGPGRTLVSSKPQAHGAPAPPSGSAPHFL.

For the purposes of the invention, the term “adrenomedullin peptide” mayrefer to any active peptide derived from the adrenomedullin precursorpeptide, unless otherwise specified. Of particular interest arehypotensive peptides. The active peptide includes, without limitation,the adrenomedullin peptide having the amino acid sequence (SEQ ID NO:6)

K(mod)GCRFGTCTVQKLAHQIYQFTDKDKDNVAPRSKISPQGY, where the lysine atresidue 1 is modified, e.g. by attachment of a lipid or other group.

In some embodiments of the invention, one or more of intermedin andadrenomedullin are modified by the methods of the invention to providefor a long-lived analog.

In some embodiments of the invention, the analogs comprise abiologically active polypeptide that has been modified at theN-terminus, a shown in structure I. In other embodiments the polypeptidemay be modified by pegylation, glycosylation, conjugation to largeproteins such as albumin, or conjugation with polymers in combinationwith amino acid modifications such as the use of D-amino acid or betaamino acids to increase the biological half-life.

where R₁ is a linear or branched C₃-C₁₀₀ alkyl; preferably a C₄-C₃₀alkyl optionally substituted with halo, hydroxy, alkoxy, amino,alkylamino, dialkylamino, sulfate, or phosphate, and which may bysaturated, or mono- or di-unsaturated, e.g. 18:0, 24:0 and 24:1. Fattyacids of interest include, without limitation, palmitic acid; stearicacid; arachidic acid; lauric acid; myristic acid; myristoleic acid;palmitoleic acid; sapienic acid; oleic acid; linoleic acid; α-linolenicacid; arachidonic acid; eicosapentaenoic acid; erucic acid;docosahexaenoic acid; etc.

Analogs of the invention provide for an in vivo effectiveness that lastsat least 2-fold longer in duration than the native peptide, and may be5-fold longer, 10-fold longer, 20-fold longer, or more. The increased invivo activity may be measured in vivo or in vitro, by determining thestability of the polypeptide, the length of the physiological effect,and the like. Of particular interest is the hypotensive effect, where asingle dose of the analog peptide effective in reducing systolic bloodpressure by at least about 10% is effective in maintaining reduced bloodpressure for at least about 1 hour, at least about 2 hours, at leastabout 3 hours, at least about 4 hours, or more.

Intermedins and adrenomedullins are ligands of the CLR/RAMP receptors,and activate the receptor upon binding. Activation by intermedin resultsin the release of prolactin, regulation of growth hormone release, inthe vascular system effects include lowering of blood pressure andvasodilation. Thus, intermedin signals through the CRLR to regulateperipheral vasodilatation-related processes. Activation byadrenomedullin results in the vascular system effects including loweringof blood pressure and vasodilation. Thus, adrenomedullin signals throughthe CRLR to regulate peripheral vasodilation-related processes.

For modification by the subject methods, native intermedin,adrenomedullin or variants thereof may be used. Peptides of interestinclude fragments of at least about 12 contiguous amino acids, moreusually at least about 20 contiguous amino acids, and may comprise 30,35, 40 or more amino acids, up to the complete peptide, and may extendfurther to comprise other sequences present in the precursor protein.Deletions may extend from residue 1 through 10 of the peptide, and mayfurther delete additionally amino acids at residues 10-15 or more.Smaller deletions, of from 1 to to 5 amino acids, may be deleted in theN-terminus. Peptides of interest for therapeutic purposes may includeall or substantially all of the provided peptide, or may comprisefragments thereof that retain the biological activity of intermedin.

The sequence of the polypeptide may be altered in various ways known inthe art to generate targeted changes in sequence. The polypeptide willusually be substantially similar to the sequences provided herein, i.e.will differ by at least one amino acid, and may differ by at least twobut not more than about ten amino acids. The sequence changes may besubstitutions, insertions or deletions. Scanning mutations thatsystematically introduce alanine, or other residues, may be used todetermine key amino acids. Conservative amino acid substitutionstypically include substitutions within the following groups: (glycine,alanine); (valine, isoleucine, leucine); (aspartic acid, glutamic acid);(asparagine, glutamine); (serine, threonine); (lysine, arginine); or(phenylalanine, tyrosine).

Modifications of interest that do not alter primary sequence includechemical derivatization of polypeptides, e.g., acetylation, orcarboxylation. Also included are modifications of glycosylation, e.g.those made by modifying the glycosylation patterns of a polypeptideduring its synthesis and processing or in further processing steps; e.g.by exposing the polypeptide to enzymes which affect glycosylation, suchas mammalian glycosylating or deglycosylating enzymes. Also embraced aresequences that have phosphorylated amino acid residues, e.g.phosphotyrosine, phosphoserine, or phosphothreonine.

Also included in the subject invention are polypeptides that have beenmodified using ordinary molecular biological techniques and syntheticchemistry so as to improve their resistance to proteolytic degradationor to optimize solubility properties or to render them more suitable asa therapeutic agent. For examples, the backbone of the peptide may becyclized to enhance stability (see Friedler et al. (2000) J. Biol. Chem.275:23783-23789). Analogs of such polypeptides include those containingresidues other than naturally occurring L-amino acids, e.g. D-aminoacids or non-naturally occurring synthetic amino acids.

The subject peptides may be prepared by in vitro synthesis, usingconventional methods as known in the art. Various commercial syntheticapparatuses are available, for example, automated synthesizers byApplied Biosystems, Inc., Foster City, Calif., Beckman, etc. By usingsynthesizers, naturally occurring amino acids may be substituted withunnatural amino acids. The particular sequence and the manner ofpreparation will be determined by convenience, economics, purityrequired, and the like.

If desired, various groups may be introduced into the peptide duringsynthesis or during expression, which allow for linking to othermolecules or to a surface. Thus cysteines can be used to makethioethers, histidines for linking to a metal ion complex, carboxylgroups for forming amides or esters, amino groups for forming amides,and the like.

The polypeptides may also be isolated and purified in accordance withconventional methods of recombinant synthesis. A lysate may be preparedof the expression host and the lysate purified using HPLC, exclusionchromatography, gel electrophoresis, affinity chromatography, or otherpurification technique. For the most part, the compositions which areused will comprise at least 20% by weight of the desired product, moreusually at least about 75% by weight, preferably at least about 95% byweight, and for therapeutic purposes, usually at least about 99.5% byweight, in relation to contaminants related to the method of preparationof the product and its purification. Usually, the percentages will bebased upon total protein.

Uses of Intermedin/Adrenomedullin

In light of the pharmacologic activities of intermedin andadrenomedullin numerous clinical indications are evident, and includewithout limitation hypertension, for example pregnancy hypertension,pulmonary arterial hypertension, hypertension associated with diabetes,etc.; bronchopulmonary dysplasia, would healing; and the like Forexample, clinical indications for which an intermedin or adrenomedullinpeptide or variants thereof may find use particularly in the treatmentof hypertension. The analogs of the invention provide for a decrease inblood pressure, e.g. systolic pressure of at least about 5%, at leastabout 10%, at least about 15%, at least about 20% or more, withoutaffecting heart rate.

Hypertension is a disease which, if untreated, strongly predisposes toatherosclerotic cardiovascular disease. It is estimated that as many as1 in 4 adult Americans have hypertension. Hypertension is approximatelytwice as common in persons with diabetes as in those without. Theprevalence of hypertension increases with age.

Hypertension should not be diagnosed on the basis of a singlemeasurement. Initial elevated readings should be confirmed on at leasttwo subsequent visits over one week or more with average diastolic bloodpressure of 90 mmHg or greater or systolic blood pressure of 140 mmHg orgreater required for diagnosis of hypertension. Special care iswarranted in diagnosing hypertension in persons with diabetes because ofgreater variability of blood pressure and a much greater likelihood ofisolated systolic hypertension. A goal blood pressure of less than130/85 mmHg is recommended for these patients.

In addition to dietary changes, pharmacological treatment may berequired to control high blood pressure. The subject peptides may beadministered to reduce arterial blood pressure. In addition, a secondaryeffect of reducing hypertension is reduction of edema and inflammatoryexudate volume.

Pharmaceutical compositions containing intermedin or adrenomedullinanalogs are useful as cardioprotective agents, e.g. to ameliorateischemic injury or myocardial infarct size consequent to myocardialischemia. The development of new therapeutic agents capable of limitingthe extent of myocardial injury, i.e., the extent of myocardialinfarction, following acute myocardial ischemia is a major concern ofmodern cardiology. There has also been interest in the development oftherapies capable of providing additional myocardial protection whichcould be administered in conjunction with thrombolytic therapy, oralone, since retrospective epidemiological studies have shown thatmortality during the first few years following infarction appears to berelated to original infarct size.

Myocardial ischemia is the result of an imbalance of myocardial oxygensupply and demand and includes exertional and vasospastic myocardialdysfunction. Exertional ischemia is generally ascribed to the presenceof critical atherosclerotic stenosis involving large coronary arteriesresulting in a reduction in subendocardial flow. Vasospastic ischemia isassociated with a spasm of focal variety, whose onset is not associatedwith exertion or stress. The spasm is better defined as an abruptincrease in vascular tone.

The compounds of this invention can be normally administered orally orparenterally, in the treatment of patients in need of cardioprotectivetherapy. The dosage regimen is that which insures maximum therapeuticresponse until improvement is obtained and thereafter the minimumeffective level that gives relief. Thus, in general, the dosages arethose that are therapeutically effective in producing a cardioprotectiveeffect, i.e., amelioration of ischemic injury or myocardial infarct sizeconsequent to myocardial ischemia. It is also anticipated that thepeptides would be useful as an injectable dosage form which may beadministered in an emergency to a patient suffering from myocardialischemia, etc.

The intermedin or adrenomedullin peptides and derivatives therefrom alsofind use in the reduction of edema, for example in rheumatoid arthritis,edema secondary to brain tumors or irradiation for cancer, edemaresulting from stroke, head trauma or spinal cord injury, post-surgicaledema, asthma and other respiratory diseases and cystoid macular edemaof the eye.

Formulations

The compounds of this invention can be incorporated into a variety offormulations for therapeutic administration. Particularly, agents thatmodulate intermedin or adrenomedullin activity, or intermedin oradrenomedullin polypeptides and analogs thereof are formulated foradministration to patients for the treatment of intermedin oradrenomedullin dysfunction, where the activity is undesirably high orlow. More particularly, the compounds of the present invention can beformulated into pharmaceutical compositions by combination withappropriate, pharmaceutically acceptable carriers or diluents, and maybe formulated into preparations in solid, semi-solid, liquid or gaseousforms, such as tablets, capsules, powders, granules, ointments,solutions, suppositories, injections, inhalants, gels, microspheres, andaerosols. As such, administration of the compounds can be achieved invarious ways, including oral, buccal, rectal, parenteral,intraperitoneal, intradermal, transdermal, intracheal, etc.,administration. The active agent may be systemic after administration ormay be localized by the use of an implant that acts to retain the activedose at the site of implantation.

In pharmaceutical dosage forms, the compounds may be administered in theform of their pharmaceutically acceptable salts, or they may also beused alone or in appropriate association, as well as in combination withother pharmaceutically active compounds. The following methods andexcipients are merely exemplary and are in no way limiting.

For oral preparations, the compounds can be used alone or in combinationwith appropriate additives to make tablets, powders, granules orcapsules, for example, with conventional additives, such as lactose,mannitol, corn starch or potato starch; with binders, such ascrystalline cellulose, cellulose derivatives, acacia, corn starch orgelatins; with disintegrators, such as corn starch, potato starch orsodium carboxymethylcellulose; with lubricants, such as talc ormagnesium stearate; and if desired, with diluents, buffering agents,moistening agents, preservatives and flavoring agents.

The compounds can be formulated into preparations for injections bydissolving, suspending or emulsifying them in an aqueous or nonaqueoussolvent, such as vegetable or other similar oils, synthetic aliphaticacid glycerides, esters of higher aliphatic acids or propylene glycol;and if desired, with conventional additives such as solubilizers,isotonic agents, suspending agents, emulsifying agents, stabilizers andpreservatives.

The compounds can be utilized in aerosol formulation to be administeredvia inhalation. The compounds of the present invention can be formulatedinto pressurized acceptable propellants such as dichlorodifluoromethane,propane, nitrogen and the like.

Furthermore, the compounds can be made into suppositories by mixing witha variety of bases such as emulsifying bases or water-soluble bases. Thecompounds of the present invention can be administered rectally via asuppository. The suppository can include vehicles such as cocoa butter,carbowaxes and polyethylene glycols, which melt at body temperature, yetare solidified at room temperature.

Unit dosage forms for oral or rectal administration such as syrups,elixirs, and suspensions may be provided wherein each dosage unit, forexample, teaspoonful, tablespoonful, tablet or suppository, contains apredetermined amount of the composition containing one or more compoundsof the present invention. Similarly, unit dosage forms for injection orintravenous administration may comprise the compound of the presentinvention in a composition as a solution in sterile water, normal salineor another pharmaceutically acceptable carrier.

Implants for sustained release formulations are well-known in the art.Implants are formulated as microspheres, slabs, etc. with biodegradableor non-biodegradable polymers. For example, polymers of lactic acidand/or glycolic acid form an erodible polymer that is well-tolerated bythe host. The implant is placed in proximity to the site of infection,so that the local concentration of active agent is increased relative tothe rest of the body.

The term “unit dosage form,” as used herein, refers to physicallydiscrete units suitable as unitary dosages for human and animalsubjects, each unit containing a predetermined quantity of compounds ofthe present invention calculated in an amount sufficient to produce thedesired effect in association with a pharmaceutically acceptablediluent, carrier or vehicle. The specifications for the novel unitdosage forms of the present invention depend on the particular compoundemployed and the effect to be achieved, and the pharmacodynamicsassociated with each compound in the host.

The pharmaceutically acceptable excipients, such as vehicles, adjuvants,carriers or diluents, are readily available to the public. Moreover,pharmaceutically acceptable auxiliary substances, such as pH adjustingand buffering agents, tonicity adjusting agents, stabilizers, wettingagents and the like, are readily available to the public.

Typical dosages for systemic administration range from 0.1 μg to 100milligrams per kg weight of subject per administration. A typical dosagemay be one tablet taken from two to six times daily, or one time-releasecapsule or tablet taken once a day and containing a proportionallyhigher content of active ingredient. The time-release effect may beobtained by capsule materials that dissolve at different pH values, bycapsules that release slowly by osmotic pressure, or by any other knownmeans of controlled release.

Those of skill will readily appreciate that dose levels can vary as afunction of the specific compound, the severity of the symptoms and thesusceptibility of the subject to side effects. Some of the specificcompounds are more potent than others. Preferred dosages for a givencompound are readily determinable by those of skill in the art by avariety of means. A preferred means is to measure the physiologicalpotency of a given compound.

Liposomes may be used for protein delivery in vivo and in vitro. Theliposomes employed in the present invention can be prepared using anyone of a variety of conventional liposome preparatory techniques. Aswill be readily apparent to those skilled in the art, such conventionaltechniques include sonication, chelate dialysis, homogenization, solventinfusion coupled with extrusion, freeze-thaw extrusion,microemulsification, as well as others. These techniques, as well asothers, are discussed, for example, in U.S. Pat. No. 4,728,578, U.K.Patent Application G.B. 2193095 A, U.S. Pat. No. 4,728,575, U.S. Pat.No. 4,737,323, International Application PCT/US85/01161, Mayer et al.,Biochimica et Biophysica Acta, Vol. 858, pp. 161-168 (1986), Hope etal., Biochimica et Biophysica Acta, Vol. 812, pp. 55-65 (1985), U.S.Pat. No. 4,533,254, Mahew et al., Methods In Enzymology, Vol. 149, pp.64-77 (1987), Mahew et al., Biochimica et Biophysica Acta, Vol. 75, pp.169-174 (1984), and Cheng et al., Investigative Radiology, Vol. 22, pp.47-55 (1987). A solvent free system similar to that described inInternational Application PCT/US85/01161 may be employed in preparingthe liposome constructions.

The materials that are utilized in preparing the liposomes include anyof the materials or combinations thereof known to those skilled in theart as suitable in liposome construction. The lipids used may be ofeither natural or synthetic origin. Such materials include, but are notlimited to, lipids such as cholesterol, phosphatidylcholine,phosphatidylethanolamine, phosphatidylserine, phosphatidylglycerol,phosphatidic acid, phosphatidylinositol, lysolipids, fatty acids,sphingomyelin, glycosphingolipids, glucolipids, glycolipids,sulphatides, lipids with amide, ether, and ester-linked fatty acids,polymerizable lipids, and combinations thereof. As one skilled in theart will recognize, the liposomes may be synthesized in the absence orpresence of incorporated glycolipid, complex carbohydrate, protein orsynthetic polymer, using conventional procedures. The surface of aliposome may also be modified with a polymer, such as, for example, withpolyethylene glycol (PEG), using procedures readily apparent to thoseskilled in the art. Any species of lipid may be used, with the soleproviso that the lipid or combination of lipids and associated materialsincorporated within the lipid matrix should form a bilayer phase underphysiologically relevant conditions. As one skilled in the art willrecognize, the composition of the liposomes may be altered to modulatethe biodistribution and clearance properties of the resulting liposomes.

The membrane bilayers in these structures typically encapsulate anaqueous volume, and form a permeability barrier between the encapsulatedvolume and the exterior solution. Lipids dispersed in aqueous solutionspontaneously form bilayers with the hydrocarbon tails directed inwardand the polar headgroups outward to interact with water. Simpleagitation of the mixture usually produces multilamellar vesicles (MLVs),structures with many bilayers in an onion-like form having diameters of1-10 .mu.m (1000-10,000 nm). Sonication of these structures, or othermethods known in the art, leads to formation of unilamellar vesicles(UVs) having an average diameter of about 30-300 nm. However, the rangeof 50 to 200 nm is considered to be optimal from the standpoint of,e.g., maximal circulation time in vivo. The actual equilibrium diameteris largely determined by the nature of the phospholipid used and theextent of incorporation of other lipids such as cholesterol. Standardmethods for the formation of liposomes are known in the art, forexample, methods for the commercial production of liposomes aredescribed in U.S. Pat. No. 4,753,788, and U.S. Pat. No. 4,935,171.

Polymerized liposomes are self-assembled aggregates of lipid molecules,and are described in U.S. Pat. Nos. 5,512,294, 6,132,764, and U.S. Pat.Application 20020071843. The hydrophobic tail groups of polymerizablelipids are derivatized with polymerizable groups, such as diacetylenegroups, which irreversibly cross-link, or polymerize, when exposed toultraviolet light or other radical, anionic or cationic, initiatingspecies, while maintaining the distribution of functional groups at thesurface of the liposome. The resulting polymerized liposome particle isstabilized against fusion with cell membranes or other liposomes andstabilized towards enzymatic degradation. The size of the polymerizedliposomes can be controlled by extrusion or other methods known to thoseskilled in the art. Polymerized liposomes may be comprised ofpolymerizable lipids, but may also comprise saturated and non-alkyne,unsaturated lipids. The polymerized liposomes can be a mixture oflipids, which provide different functional groups on the hydrophilicexposed surface. For example, some hydrophilic head groups can havefunctional surface groups, for example, biotin, amines, cyano,carboxylic acids, isothiocyanates, thiols, disulfides, α-halocarbonylcompounds, α,β-unsaturated carbonyl compounds and alkyl hydrazines.These groups can be used for attachment of nucleic acid sequences.

For use in the above described formulations, intermedin oradrenomedullin or derivatives therefrom may be synthesized and stored asa solid lyophilized powder which is reconstituted into apharmaceutically acceptable liquid immediately prior to use. Suchformulations are usually preferred because it is recognized by thoseskilled in the art that lyophilized preparations generally maintainpharmaceutical activity better over time than their liquid counterparts.

In addition, intermedin or adrenomedullin and their analogs could beapplied topically on the skin as well as administered as aerosal sprays.

Alternatively, the peptides may be formulated as a liquid, e.g.comprising a buffer at a concentration of from about 1 mM to about 50 mMthat functions to maintain the pH, wherein the anion of said buffer maybe selected from the group consisting of acetate, phosphate, carbonate,succinate, citrate, borate, tartrate, fumarate and lactate; and analcohol which may be selected from the group consisting of mannitol,sorbitol, ribotol, arabitol, xylitol, inositol, galactitol, methanol,ethanol and glycerol. Other additives may include amino acids such asmethionine, arginine, lysine, glutamic acid, cysteine, glutathione, andthe like, where amino acids are generally present in concentrationsranging from about 1 mM to about 100 mM. Various sugars are optionallyincluded in the formulations, including, for example, glucose, sucrose,lactose, fructose, trehalose, mannose, and the like. Additive sugars aregenerally present in concentrations ranging from about 1% to about 10%.

EXPERIMENTAL

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the present invention, and are not intended to limit thescope of what the inventors regard as their invention nor are theyintended to represent that the experiments below are all or the onlyexperiments performed. Efforts have been made to ensure accuracy withrespect to numbers used (e.g., amounts, temperature, etc.) but someexperimental errors and deviations should be accounted for. Unlessindicated otherwise, parts are parts by weight, molecular weight isweight average molecular weight, temperature is in degrees Centigrade,and pressure is at or near atmospheric.

Example 1

Modification of Intermedin.

Peptides were synthesized on an Applied Biosystems automated peptidesynthesizer by the Pan Facility at Stanford University using standardsolid-phase Fmoc peptide chemistry (Fields G B, Noble R L. Solid phasepeptide synthesis utilizing 9-fluorenylmethoxycarbonyl amino acids. IntJ Pept Protein Res 35: 161-214, 1990). The modified peptides wassynthesized by incorporating lysine residue that has been conjugated toan Fmoc-protected [C16]palmitate fatty acid (Lys(PAL)) during thesynthesis of peptides. Purity was determined by reversed-phase HPLC andsubsequently characterized using electrospray ionisation massspectrometry.

The in vivo biological activity of modified and native intermedin in asset forth in FIGS. 1-4. The intermedin peptides were dissolved in salinesolution with 10-20% DMSO at 10 micromoles/liter, Before injection,aliquots of peptides were dissolved in PBS to a injection final volumeof 100 ul. Blood pressure measurements were made in conscious SHR rats(9-16 weeks of age) pre-adapted to the measurement procedure. Indirectsystolic pressure was determined by a programmable non-invasive bloodpressure system using the tail-cuff method (Kent ScientificCorporation). Following attachment of the pressure transducer, rats wereleft undisturbed for 10 min before base-line measurements that spanned a15-20 minute interval. Following base-line measurements, rats wereinjected intraperitoneally with varying doses of peptides, or salinewith 10% DMSO. Blood pressure and heart rate were monitored for 20-40min at 30-s intervals. Changes in blood pressure were calculated as theaverage of measurements performed within a given time interval. Thebasal blood pressure of control animals are comparable to those treatedwith the intermedin peptide.

As shown in FIGS. 1A-1C, when rats are injected with the modifiedintermedin (IMD-Palmitic acid-modified IMD) and blood pressure ismeasured over time, there is a significant and long lasting drop insystolic blood pressure. The hypotensive effects of this peptide lastedover five hours in vivo, whereas that of the unmodified IMD peptidelasted less than 1 hr. The results from control animals injected withPBS is depicted in FIG. 3, and from the unmodified peptide in FIG. 2.

Example 2

Modification of Adrenomedullin.

Peptides were synthesized on an Applied Biosystems automated peptidesynthesizer by the Pan Facility at Stanford University using standardsolid-phase Fmoc peptide chemistry (Fields G B, Noble R L. Solid phasepeptide synthesis utilizing 9-fluorenylmethoxycarbonyl amino acids. IntJ Pept Protein Res 35: 161-214, 1990). The modified peptides wassynthesized by incorporating lysine residue that has been conjugated toan Fmoc-protected [C16]palmitate fatty acid (Lys(PAL)) during thesynthesis of peptides. Purity was determined by reversed-phase HPLC andsubsequently characterized using electrospray ionisation massspectrometry.

Biological activity of adrenomedullin in vitro. A typical demonstrationof the stimulatory effect of ADM and ADM-PA is shown in FIG. 7.

Biological activity of adrenomedullin analog in vivo. The adrenomedullinpeptides were dissolved in saline solution with 10-20% DMSO at 10micromoles/liter, Before injection, aliquots of peptides were dissolvedin PBS to a injection final volume of 200 μl. Blood pressuremeasurements were made in conscious SHR rats (9-16 weeks of age)pre-adapted to the measurement procedure. Indirect systolic pressure wasdetermined by a programmable non-invasive blood pressure system usingthe tail-cuff method (Kent Scientific Corporation). Following attachmentof the pressure transducer, rats were left undisturbed for 10 min beforebase-line measurements that spanned a 10-15 minute interval. Followingbase-line measurements, rats were injected intraperitoneally withvarying doses of peptides, or saline with 10% DMSO. Blood pressure andheart rate were monitored for 20-40 min at 20-s intervals. Changes inblood pressure were calculated as the average of measurements performedwithin a given time interval.

The data show the systolic blood pressure profile of SHR rats afterinjection of the ADM-PA peptide or saline solution. (see Roh et al. MolEndocrinol. 2005 November; 19 (11):2824-38).

As shown in FIGS. 5A-5C, when rats are injected with 100 nmoles/kg bodyweight of the modified adrenomedullin (ADM-Palmitic acid-modified ADM)and blood pressure is measured over time, there is a significant andlong lasting drop in systolic blood pressure. The ADM-PA peptide has along half-life as compared to ADM using based on measurement ofimmunoreactive ADM using ELISA (FIG. 8).

All publications and patent applications cited in this specification areherein incorporated by reference as if each individual publication orpatent application were specifically and individually indicated to beincorporated by reference.

The present invention has been described in terms of particularembodiments found or proposed by the present inventor to comprisepreferred modes for the practice of the invention. It will beappreciated by those of skill in the art that, in light of the presentdisclosure, numerous modifications and changes can be made in theparticular embodiments exemplified without departing from the intendedscope of the invention. Moreover, due to biological functionalequivalency considerations, changes can be made in protein structurewithout affecting the biological action in kind or amount. All suchmodifications are intended to be included within the scope of theappended claims.

What is claimed is:
 1. A method of treating hypertension, the methodcomprising: administering an effective dose of a biologically activehuman adrenomedullin peptide modified by conjugation of a fatty acid tothe amino terminus of the peptide, wherein the modified peptide has aserum half-life of greater than 1.5 hours, and wherein theadrenomedullin peptide consists of the amino acid sequence set forth asSEQ ID NO:6.
 2. The method of claim 1, wherein the peptide reducessystolic blood pressure by at least about 10% without increasing heartrate.
 3. The method of claim 1, wherein the hypertension is pregnancyhypertension.